Nanoparticle Drug Delivery to Retinal Ganglion Cells in Glaucoma G.C. Shih1, M.G. Sternberg1, S.D. Crish1, E.M. Harth2, D.J. Calkins1

1Vanderbilt Eye Institute, 2Vanderbilt Department of Chemistry

Vanderbilt University Medical Center, Nashville, TN

.

Purpose

Methods

Patient Compliance (IOP data)

RGC Uptake

We constructed a 53nm nanosponge (Reti-Nano) with encapsulated DiO to measure retinal deposition following intravitreal injection in C57BL/6 mice. In addition, RGC DiO uptake was examined in phosphorylated heavy-chain neurofilament (SMI31)+ RGCs. To determine the efficacy of brimonidine- and travatan-loaded nanosponges in lowering IOP, C57BL/6 mice underwent microbead injection into the anterior chamber of both eyes to induce IOP elevation. Mice then received a single intravitreal injection of brimonidine- or travatan-loaded nanoparticles into one eye and PBS in the other. IOP was measured in both eyes up to 10 days post-injection.

Current marketed treatments for glaucoma include surgical alteration of eye structure and

medications administered topically, all of which aim to either improve aqueous drainage

or decrease aqueous production. However, current glaucoma treatments are problematic

for a variety of reasons, one of the largest problems being patient compliance. Also,

lowering IOP does not necessarily stop neurodegeneration, thus prompting exploration of

neuroprotective agents as treatments for glaucoma. The particular success of topical

neuroprotective agents has been variable, as drug delivery to the RGCs in the posterior

chamber of the eye is problematic.

Here we examined the feasibility of using nanoparticle-encapsulated agents administered

intravitreally in mice with elevated ocular pressure.

Ideally, the creation of an optimal retinal drug delivery system would provide more

consistent therapeutic outcomes and increase treatment success rates.

Conclusion

Reti-Nano nanoparticles reached the retina, remained there for a period of

several weeks, and showed no obvious toxic effects. The presence of

nanoparticles in the vitreal chamber, extracellular space, and intracellularly for

days to weeks enhances their long-term retinal drug delivery potential. DiO

localization to the nerve fiber layer and uptake by RGCs indicates that the

nanoparticles passed the inner limiting membrane, a major barrier to many

therapeutic compounds. Together with the reduction of IOP by nanoparticle-

encapsulated brimonidine or nanoparticle-encapsulated travatan, this data

suggests intravitreal injections of therapeutically-loaded nanoparticles may be

useful as a long-term drug delivery system in glaucoma.

Results

• The retina appeared free of pathology at all time points after NP injection.

• Retinal DiO deposition increased 3-fold after 14 days and 8-fold after 28 days

compared to baseline.

• About 40% of SMI31+ RGCs also contained DiO.

• Travatan NP treatment resulted in a reduction of elevated IOP to near baseline

levels over the course of 5 days.

• Brimonidine NP treatment also resulted in a reduction of elevated IOP near

baseline levels over the course of 10 days.

Travatan- and Brimonidine-Loaded Nanoparticle

Injection Treatments Reduce IOP to Near Baseline Levels

Sappington et al.: IOVS. 2010

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Evidence of DiO Uptake by Retinal Ganglion Cells

Retinal Deposition of DiO Increased Over Time

Left: Confocal

micrographs of

whole mount retina

showing

accumulations of

NPs and RGCs

stained for SMI31.

Individual RGC

uptake of DiO

molecules is

apparent (circles).

DiONP

0.1 mm

SMI31 + DiONP

0.1 mm

DiONP SMI31 DiONP + SMI31

25 µm

Top Left: Microbead injection into the anterior chamber of a rat eye

using a glass micropipette (approximately 100-μm diameter).

Top Center: Vertical section of flat-mounted anterior chamber from

an eye injected with 5 μL microbeads. Microbeads are apparent in

the iridocorneal angle and cluster near the point of aqueous outflow

(arrow).

Top Right: Photomicrograph of the anterior segment immediately

after injection with 5 μL microbeads.

Bottom Right: Photomicrographs of the anterior segment

immediately after injection with 5 μL saline.

. Multiple copies of

linear polyester

Controlled equivalents

of cross-linking unit

Intermolecular chain cross-linking

Above: Reti-Nano nanoparticle sponge structure

DESCRIPTION OF RETI-NANO

• A 53 nm NP that releases drug (or other

payload) over time as it degrades.

– Prepared from linear polyester

precursors coupled with cross-linking

units.

– By varying the amount of cross-linker

per polyester group, NPs with distinct

nanoscopic dimensions can be formed

(van der Ende et al. 2008 & 2009).

• Advantages of Reti-Nano

– NPs have amorphous properties at 37°C

that positively contribute to a linear

degradation and a controlled release of

therapeutics without a “burst effect”.

– Exhibit an increased drug load capacity

(3X greater than traditional polyester

particles).

(van der Ende et al.: J. Am. Chem. Soc. 2008)

Left: Hydrolytic degradation studies of (▲) 725.1 ± 94.3 nm AB1 nanoparticles; (◼) 115.6 ± 12.5

nm AB1 nanoparticles; and () 30.71 ± 2.21 nm AB1 nanoparticles.

Right: In vitro release profile of paclitaxel from particles loaded with 11.3% paclitaxel prepared

with the emulsification process. The cumulative release profile shows a desirable controlled and

sustained release of paclitaxel from the nanoparticles (van der Ende et al.: Polym. Chem. 2010).

0

10

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30

40

50

60

0 5 10 15 20 25 30

Ret

inal

Su

rface

Cover

ed (

%)

Days Post-Treatment

Above: Confocal fluorescent

micrographs of whole-mount

retina immunostained for

phosphorylated heavy chain

neurofilament (SMI31) and

demonstrating DiO deposition.

Right: With an increase in

time following NP injection,

there is a linear increase in the

percentage of retinal surface

covered by DiO deposition.

Subjective Retinal Coverage vs. Time

Topical Travatan vs Intravitreal Travatan Nanoparticles

Days

0 2 4 6 8 10 12 14

Intr

ao

cu

lar

Pre

ssu

re (

mm

Hg

)

12

14

16

18

20

22

24

A B C

A: Bilateral microbead injectionB: 1st topical Travatan or intravitreal nanoparticle injectionC: 2nd topical Travatan

Topical Travatan

Intravitreal Travatan Nanoparticles

Intravitreal Lumigan Nanoparticles vs Control

Days

0 5 10 15 20 25 30

Intr

ao

cu

lar

Pre

ssu

re (

mm

Hg

)

10

12

14

16

18

20

22

24Intravitreal PBS

Intravitreal Lumigan nanoparticles

A B A: Bilateral microbead injectionB: Intravitreal injections

Intravitreal Lumigan Nanoparticles vs Control

Days

0 10 20 30 40 50

Intr

ao

cu

lar

Pre

ssu

re (

mm

Hg

)

10

12

14

16

18

20

22

24Intravitreal PBS

Intravitreal Lumigan nanoparticles

A B A: Bilateral microbead injectionB: Intravitreal injections

Cornea

Iris

Ciliary Body